The discovery that fetal hemoglobin can ameliorate the clinical severity of sickle cell disease generated considerable interest in the reactivation of the silenced gamma-globin genes in adult life as a potential therapeutic approach in patients with this disease. Intensive investigation of the molecular bases of the genetic disorders that are characterized by the persistence of fetal hemoglobin production in adult life has not translated into effective therapies. In contrast, the study of epigenetic modifications (e.g. DNA methylation and histone acetylation) in the developmental silencing of the gamma-globin genes spurred the development of a number of therapeutic agents that can induce fetal hemoglobin and ameliorate the severity of sickle cell disease. For the past 8 years, we have been conducting clinical trials of butyrate and hydroxyurea in sickle cell disease. In the course of these studies, we made a number of important and at times unexpected clinical observations that raised new questions about the mechanism(s) of induction of fetal hemoglobin by these agents. The major aim of this application is to study the potential role of the epigenetic state of the beta-globin gene cluster in the variable silencing of the gamma-globin genes and in the reactivation of fetal hemoglobin by butyrate and hydroxyurea. The specific aims are to: 1) Compare the state of histone acetylation and DNA methylation in the gamma-globin gene cluster of patients with variable silencing (i.e. either complete or partial silencing) of the beta-globin genes; 2) Investigate the effects of butyrate and hydroxyurea on histone acetylation and DNA methylation in the beta-globin gene cluster; 3) Investigate the potential roles of histone acetylation and DNA methylation in butyrate resistance and the reversal of this resistance by pre-treatment with hydroxyurea; 4) Investigate the heritability of the induction of gamma-globin gene expression by butyrate and nonbutyrate inhibitors of histone deacetylases; 5) Investigate the effects of butyrate and other inhibitors of histone deacetylase on the translational efficiency of gamma-globin mRNA. We believe these studies will shed important light on the normal mechanisms of silencing of the gamma-globin genes and their reversal by pharmacological therapies.